DNA genotyping is a process of determining the sequence of DNA nucleotides at a generic locus, or at a position on a chromosome of a gene or other chromosome marker. For the purpose of identifying a human, certain generic loci have been selected as the standard markers to characterize the DNA. Each marker is a DNA fragment containing a repetition of a certain nucleotide sequence. Generally, there are thirteen (13) cores and several other accepted standard markers. These markers contain short repetitions (e.g., roughly from five (5) to forty (40)) of four (4) nucleotides. They are in the class of Short Tandem Repeat (STR) of DNA sequence.
The repetition numbers at these markers varies rather randomly from person to person. The specific form of DNA sequence at a generic locus is called an allele, which provides sufficient differentiation among people. The STR sequence is inherited from the parents DNA. At each marker, there may be two different alleles, one from each parent, and the marker is referred to as heterozygous. If the alleles from both parents have same STR numbers, the marker is referred to as homozygous. If the alleles of thirteen (13) core markers were heterozygous, each person would have twenty (26) different allele numbers. Assume each number is evenly distributed over a range of ten (10), the likelihood of having two people with the same alleles numbers from these thirteen (13) markers is extremely small.
To measure allele numbers, a DNA fragment containing all STR nucleotides and adjacent sections of nucleotides at each locus is copied from the DNA sample, and replicated by a technique called polymerase chain reaction (PCR). The fragment size is measured in the unit of base pairs, where a base pair is the size of a pair of DNA nucleotides. A substance containing synthesized fragments of known fragment sizes is added to the sample fluid. These added fragments are measured simultaneous with the DNA fragments. Because the sizes of these added fragments are known, they provide the means to calibrate for the DNA fragment sizes, and they have been referred to as internal sizing standards or internal lane standard (ILS) fragments.
A modern apparatus for DNA analysis uses a rigid sample carrier called biochip which contains multiple capillaries in parallel to run multiple samples simultaneously. The sample fluid is injected into one end of the capillary. The capillary has a negative electrode at injection end and positive electrode at other end. When a high voltage is applied to the electrodes, the electric field exerts a net electrostatic force on the surface charge of the fragment. The fragment moves or migrates toward the positive electrode at a speed depends on the electric field strength, the fragment size, and other factors. In this electrophoresis process, fragments of the same size arrives the positive electrode end at the same time, and they are separated from other fragments of different sizes.
The sizes of the fragments in a DNA locus are known to be within certain range. It is possible to find a number of loci in which the fragment sizes of a locus do not overlapped with other loci. Furthermore, it is possible to divide the whole set of loci into several groups. In each group, the fragment sizes of a locus are separated from other loci, and it is called a color group. For each color group, a dye with a distinct fluorescent color is attached to the fragments of all loci in the group. Usually, the dye is attached to a molecule called primer at one end of the fragment.
The fragments are separated by the electrophoresis process and detected by an optical system as a digital signal. A fragment is detected as a peak in the signal. The detection time (or the acquisition time) of peak can be used to determine the fragment size. Based on the non-overlapping range of the loci in the color group, the measured fragment size identifies the locus of the fragment. With other supporting data, the measured fragment size can be used to identify it as one of DNA fragments in the locus with known STR number.
The sample is prepared with multiple dyes with one dye for each color group. When the sample is excited by the light source, the fluorescent light is mixed with multiple colors from these dyes. It is necessary to use optical filter to separate the fluorescent colors. Each filtered fluorescent color is measured in a detection channel as an electrical signal. The strength of the signal is proportional to the amount of the fragments in the fluid. The ILS substance is labeled with a color different from the colors of the STR markers, such that the ILS signal can be separated from the signals of the markers.
To increase the measurement throughput, most of the apparatus is designed with capability to run multiple capillaries simultaneously, each containing a DNA sample of interest. These capillaries are packed into one biochip. Each capillary is referred to a “lane” or “sample channel” of the biochip. Advanced apparatus may use a biochip made of durable transparent glass or plastic and contain multiple capillaries with diameter as small as hundred (100) microns or less. The biochip is intended to be disposable, with one biochip used once only for one run.
In reality, the preparation and the environment of the sample fluid are not perfect. The migration speed of the fragments in the electrophoresis varies from one lane to another lane. It further varies from one run to another run. The migration times between fastest and slowest lanes can possibly differ by as much as a factor of two (2). As the result, the fragment sizes cannot be determined simply from the acquisition time of the peaks in the signal. The ILS substance contains only fragments of known sizes. These fragments migrate through the capillary under the same condition.
The acquisition times of the peaks in the ILS signal can be used as the scale to translate the acquisition time of a sample fragment into an ILS-translated fragment size. Unfortunately, the migration speed of a DNA fragment depends slightly on its nucleotide sequence and other factors. Consequently, the fragment size measured by the ILS signal (the ILS-translated fragment size) does not measure exactly the actual DNA fragment size, unless the DNA fragment happen to have the same nucleotide sequence as the ILS fragments.
To correct for this difference, a substance that contains virtually all possible DNA fragments of the markers is prepared and processed in a dedicated lane. The signal of this substance is called allelic ladder signal. The allelic ladder substance is prepared by combining genomic DNA or locus-specific PCR products from multiple people in a population. Each fragment in the ladder is considered a genuine DNA fragment, and its DNA fragment size is known and stays constant. The allelic ladder signal is detected like the DNA sample in multiple colors. Each peak in the allelic ladder signal indicates the expected peak position of a DNA fragment.
The ILS-translated fragment size for each of the ladder fragments is measured. Based on that, the allelic ladder signal provides a way to map a sample fragment from ILS-translated fragment size into actual DNA fragment size. In fact, for the allele number determination purpose here, the actual DNA fragment size is not important. The importance is the allele number of a DNA sample fragment. The allele number of every true ladder signal peak is known. Thus, the allelic ladder signal provides a way to map a sample fragment from ILS-translated fragment size into allele number or vice versus.